Antimicrobial coatings

ABSTRACT

The present invention provides a substrate having a plurality of immobilised furanone moieties associated with at least part of a surface of the substrate. The invention also relates to articles consisting of or comprising such a substrate.

FIELD OF THE INVENTION

[0001] The present invention relates to substrates having one or morebioactive species immobilized on a surface thereof. The presentinvention is particularly concerned with substrates having anantibacterial or antifungal species immobilized thereon and to methodsof forming such substrates.

BACKGROUND

[0002] The present invention will now be described with particularreference to biomedical applications. However, it is to be understoodthat the present invention has applications in any area that requiresincreased resistance to bacterial and fungal colonization.

[0003] The colonization by bacteria of devices used for human healthcare and/or improvement of quality of life poses serious problems andcauses adverse reactions that are detrimental to the viability anduseful service life of the device. Examples of devices that arecolonized by bacteria comprise implantable biomedical devices such asurinary catheters, percutaneous access catheters, stents, as well asnon-implantable devices such as contact lenses, contact lens storagecases, and the like. Once attached to a surface, bacteria are much moreresistant to antibiotics and other bacteriostatic and bacteriocidalagents and can proliferate, inducing adverse reactions in the hostenvironment.

[0004] Accordingly, much work has focused on the prevention of bacterialcolonization. One solution is to coat catheters with a thin layer ofsilver metal, which releases silver ions that act as antibacterialagents. Another solution is the surface immobilization of quaternaryamine compounds (Dziabo U.S. Pat. No. 5,515,117) which are known to beantibacterial agents.

[0005] The provision of silver coatings is not practical or economic inmany applications. There is also clinical evidence that silver coatingsdo not provide adequate effectiveness in all desirable circumstances.Quaternary amine compounds likewise possess shortcomings in that theycan induce cell toxicity with host cells, which adversely affects thecontinued viability of fully functioning tissues adjacent to thebiomedical implant. While the cytotoxicity of quaternary amine compoundsis relatively mild, it is possible to observe in in vitro cell cultureexperiments that the shape and biological functions of cells in contactwith such compounds are substantially affected.

[0006] Furanone compounds have been reported to be effective agentsagainst bacterial proliferation and to have antifungal properties (seefor example Reichelt and Borowitzka (1984) Hydrobiologia 116: 158-168and International Patent Application Nos. PCT/AU99/00284 andPCT/AU96/00167, the disclosures of which are incorporated herein byreference). They are thought to act by interfering with bacterialproperties that are regulated by acylated homoserine lactones, (AHLs)and two component phosphorelay signal transduction systems. These arefundamental regulatory agents which are widespread in bacteria,including human pathogens (see for example International PatentApplication Nos. PCT/AU96/00167 and PCT/AU00/01553, the disclosures ofwhich is incorporated herein by reference).

[0007] The AHL regulatory systems in bacteria are one type of signaltransduction systems which regulate intercellular activity in responseto environmental conditions and extracellular signal molecules. Thissystem was first discovered in the bioluminescent mane bacteria Vibrioharveyi and V. fischeri where it is used to control expression ofbioluminescence. In principle, the system is comprised of twoproteins—LuxR and LuxI. The LuxI enzyme is encoded by a luxI gene andproduces a related family of signal molecules known as the acylatedhomoserine lactones (AHLs). These signal molecules bind to the LuxRregulator which is then activated and serves both as a positiveregulator for the structural genes which encode the enzymes responsiblefor bioluminescence, and as a positive regulator for the luxI geneitself. The entire system is amplified via a process of auto induction.Additional molecules serve as regulators of the LuxR-LuxI system.

[0008] While initially discovered for bioluminescent bacteria, thisregulatory system has now been found in numerous other microorganisms,and is involved in a wide variety of bacterial activities (Pesci andIglewski, 1999, In Cell-cell signaling in bacteria. Dunny and Winans(eds), ASM Press, Washington D.C., Stevens and Greenberg, 1999 InCell-cell signaling in bacteria. Dunny and Winans (eds), ASM Press,Washington D.C., Pierson et al. 1998, Annu. Rev. Phytopathol.36:207-225). These activities include, but are not restricted toexoenzyme production in the plant pathogen Erwinia carotovora andexoenzyme and virulence factor production in Pseudomonas aeruginosa, thecausative agent of cystic fibrosis, and Ti plasmid transfer fromAgrobacterium tumefaciens to plants. In all instances acylatedhomoserine lactone, or homoserine lactone-like compounds are theregulatory autoinducers.

[0009] Two-component phosphorelay signal transduction systems representanother mechanism, which is distinct from the AHL system describedabove, by which bacteria sense and respond to their environment (seePCT/AU00/01553). Two-component transduction systems play important rolesin the growth and maintenance and functionality of many differentmicroorganisms. Examples include, but not are limited to, regulation ofthe production of exopolysaccharides and virulence factors; theregulation of motility, swarming, attachment and biofilm formation; andmaintenance of viability.

[0010] Since these regulatory systems are widespread among bacteria andbecause they control processes leading to bacterial invasion of hostorganisms, it is likely that other organisms will have evolved defencemechanisms against these systems.

[0011] Natural furanones and their synthetic analogues have been shownto inhibit bacterial adhesion (PCT/AU96/00167). The presumed mode ofaction of interfering with the regulation of AHL and two componentphosphorelay systems entails that the compounds should be capable ofdiffusing into and through the bacterial cell in order to reach thetarget site. As a result, soluble, low-molecular weight furanones havebeen used to date as antibacterial agents.

[0012] However, surprisingly, we have found that furanone compoundsimmobilized onto polymeric substrate surfaces by stable covalent bondsstill maintain antibacterial activity, in preventing bacterialproliferation on that substrate material. This surprising finding is atodds with the presumed intracellular action of furanone compounds as AHLmimics, and their ability to interfere with signal transduction throughthe two-component phosphorelay systems and cannot be explained atpresent with a well-supported mechanistic model.

DESCRIPTION OF INVENTION

[0013] Accordingly, in a first aspect, the present invention provides asubstrate having a plurality of immobilised furanone moieties associatedwith at least part of a surface of the substrate.

[0014] By “associated with at least part of a surface of the substrate”is meant immobilization directly onto at least part of the surface ofthe material of the substrate or via one or more intermediate layersinterposed between the substrate material and the immobilised layer. Theintermediate layer(s) may be bonding layer(s).

[0015] By the term “furanone moiety” is meant a moiety derived from afuranone compound or an analog of the furanone compound or a combinationof two or more furanone compounds.

[0016] The furanone moiety may be derived from a natural or syntheticfuranone compound.

[0017] The furanone compound is preferably a compound of formula:

[0018] wherein R₁ is a moiety selected from the group consisting of H,halogen, formyl, carboxyl, cyano. ester, amide, alkyl, alkoxy, oxoalkyl,alkenyl, alkynyl, aryl or arylalkyl, which moiety may optionally be

[0019] substituted by one or more substituents; and/or

[0020] interrupted by one or more hetero atoms; and/or

[0021] straight chain, branched chain, hydrophobic, hydrophilic orfluorophilic;

[0022] R₂, R₃ and R₄ are independently or all H or halogen;

[0023] and “═” represents either a double bond or a single bond.

[0024] In the formula, a particular geometry is not to be taken asspecified. For example, the formula covers both Z- and E-isomers.

[0025] Examples of suitable furanone compounds are those disclosed inInternational Patent Application Nos. PCT/AU95/00407, PCT/AU96/00167,PCT/AU98/00508 and PCT/AU99/00285, the entire disclosures of which areincorporated herein by cross-reference.

[0026] The immobilized furanone moieties may be derived from onefuranone compound or a plurality of different furanone compoundsselected, for example, for both their antibacterial activity and absenceof cytotoxicity as well as any other adverse biomedical effect on thehost environment that the coated substrate contacts.

[0027] The substrate may be shaped or non-shaped. The substrate may besolid, semi-solid or flexible. The substrate may be a woven or non-wovenfilm or sheet. The substrate may be a natural or synthetic filament orfibre. The substrate may be a natural material, for example, a plantseed. The material from which the substrate is formed may be selected tosuit the particular application. For example, in the case of a shapedbiomedical device the material may meet other specifications of theapplication, such as mechanical and optical properties.

[0028] The invention, in a second aspect, includes an article consistingof or including a substrate in accordance with the first aspect of theinvention.

[0029] Examples of articles include, but are not limited, to implantablebiomedical devices such as urinary catheters, percutaneous accesscatheters, stents, as well as non-implantable devices such as contactlenses, contact lens storage cases, and the like.

[0030] The material from which the article is formed can be a metal, aceramic, a solid synthetic polymer, or a solid natural polymer, forexample a solid biopolymer. Examples of useful materials for thisinvention are titanium, hydroxyapatite, polyethylene (which are usefulmaterials for orthopaedic implants), polyurethanes, organosiloxanepolymers, perfluorinated polymers (which are useful materials forinstance for catheters, soft tissue augmentation, and blood contactingdevices such as heart valves), acrylic hydrogel polymers and siloxanehydrogel polymers (for instance for contact lens and intraocular lensapplications), and the like, and any combination thereof. The surfacesof these materials can be chemically inert or contain reactivefunctional groups.

[0031] In this specification the term “substituted” means that a groupmay or may not be further substituted with one or more groups selectedfrom alkyl, cycloalkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkynyl,hydroxy, alkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino,nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino,dialkylamino, alkenylamine, alkynylamino, acyl, alkenacyl, alkynylacyl,acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocyclyl,heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulfenyl,carboalkoxy, alkylthio, acylthio, phosphorus-containing groups such asphosphono and phosphinyl.

[0032] The term “alkyl”, used either alone or in compound words such as“haloalkyl” or “alkylthio”, denotes straight chain or branched C₁₋₆alkyl groups. Examples include methyl, ethyl, propyl, isopropyl and thelike.

[0033] The term “alkoxy” denotes straight chain or branched alkoxy,preferably C₁₋₁₀ alkoxy. Examples include methoxy, ethoxy, n-propoxy,isopropoxy and the different butoxy isomers.

[0034] The term “alkenyl” denotes groups formed from straight chain,branched or mono- or polycyclic alkenes including ethylenically mono- orpoly-unsaturated alkyl or cycloalkyl groups as previously defined,preferably C₂₋₁₀ alkenyl. Examples of alkenyl include vinyl, allyl,1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl,cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl,cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl,1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl,1-4,pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl,1,3,5-cycloheptatrienyl,or 1,3,5,7-cyclooctatetraenyl.

[0035] The term “halogen” denotes fluorine, chlorine, bromine or iodine,preferably bromine or fluorine.

[0036] The term “heteroatoms” denotes O, N or S.

[0037] The term “acyl” used either alone or in compound words such as“acyloxy”, “acylthio”, “acylamino” or diacylamino” denotes carbamoyl,aliphatic acyl group and acyl group containing a heterocyclic ring whichis referred to as heterocyclic acyl, preferably C₁₋₁₀ acyl. Examples ofacyl include carbamoyl: straight chain or branched alkanoyl, such asformyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl,2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,decanoyl; alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl,t-butoxycarbonyl, t-pentyloxycarbonyl or heptyloxycarbonyl;cycloalkylcarbonyl such as cyclopopylcarbonyl cyclobutylcarbonyl,cyclopentylcarbonyl or cyclohexylcarbonyl; alkylsulfonyl, such asmethylsulfonyl or ethylsulfonyl; alkoxysulfonyl, such as methoxysulfonylor ethoxysulfonyl; heterocyclylcarbonyl; heterocyclylalkanoyl, such aspyrrolidinylacetyl, pyrrolidinylpropanoyl, pyrrolidinylbutanoyl,pyrrolidinylpentanoyl, pyrrolidinylhexanoyl or thiazolidinylacetyl;heterocyclylalkenoyl, such as heterocyclylpropenoyl,heterocyclylbutenoyl, heterocyclylpentenoyl or heterocyclylhexenoyl; orheterocyclylglyoxyloyl, such as, thiazolidinylglyoxyloyl orpyrrolidinylglyoxyloyl.

[0038] The term “fluorophilic” is used to indicate the highly attractiveinteractions certain groups, such as highly fluorinated alkyl groups ofC4-C10 chain length, have for perfluoroalkanes and perfluoroalkanepolymers.

[0039] The present invention also has application in any article innon-biomedical areas that requires increased resistance to bacterialcolonization. For example, female hygiene products and food preparationand storage ware. In each case the nature and properties of thesubstrate are selected for that purpose.

[0040] As some furanone compounds also possess antifungal properties,the process of surface coating by the process of this invention may alsobe used to protect materials and devices from fungal attack. Examplesinclude archival documents, antiques and art, rare and valuable seedsintended for storage (e.g. seed banks of conservation groups), etc inwhich case the substrate may be paper, material or other natural orsynthetic material.

[0041] In a third aspect, the present invention provides a method offorming an antimicrobial and/or antifungal layer on a substrateincluding:

[0042] (a) providing a substrate;

[0043] (b) providing at least one furanone compound or an analogthereof;

[0044] (c) optionally treating at least part of the surface of thesubstrate to activate the surface;

[0045] (d) reacting the at least one furanone compound or an analogthereof; with the optionally treated surface to immobilize the at leastone furanone or analog.

[0046] Reference to at least part of the surface of the substrateincludes a surface of one or more intermediate layers applied to thesubstrate.

[0047] The furanone compound(s) may be immobilised on the substratesurface by any suitable technique. Immobilization may be by covalent ornon-covalent means. Preferably, the furanone compounds are immobilizedon the substrate surface by means of covalent bonds.

[0048] Accordingly, in a fourth aspect the present invention provides asubstrate according to the first aspect wherein the furanone moietiesare covalently bonded to a surface of the substrate.

[0049] The immobilization of furanone compounds on to the substrateprevents their loss from the surface, thus ensuring long-lastingantimicrobial action.

[0050] The substrates in accordance with the present invention may becharacterised by the formula:

X—Y—Z

[0051] where X is a substrate, Y is an optional chemical King moiety andZ is a furanone moiety. The linking moiety, if present, may be ahomobifunctional or heterobifunctional linking moiety. Y may be a simplecomponent (eg a short molecule) or it may comprise a plurality of unitsor components that may be the same of different Y may comprise a numberof components or units that may be “built up” in a stepwise fashion.

[0052] The formation of a covalent interfacial linkage is muchpreferable to an ionic bond since in biological media where the saltcontent is such that ionic bonds are interfered with and ironicallyattached molecules can be displaced from a surface.

[0053] The covalent anchoring of the furanone compound(s) also serves toeliminate concerns regarding possible deleterious effects that furanonecompounds might cause at sites distant from the biomedical device, suchas in the liver, brain, or kidney tissues of a living human organism. Inmedical applications it is important to anchor the furanone compound(s)via an interfacial covalent bond that is not subject to cleavage in thebiomedical host environment that the biomedical device is to be placedin.

[0054] Methods for the covalent immobilization of organic molecules ontosolid surfaces are well known to those skilled in the art Interfacialreactions leading to the formation of covalent interfacial bonds arederived from well-known organic-synthetic reactions. The choice ofimmobilization reaction depends on both the nature of the substratematerial and the chemical composition of the furanone derivative(s) thatare desired for a particular application.

[0055] For example, a furanone derivative that contains a hydroxyl groupin a side chain distal to the furanone ring system, can be linkedcovalently onto surfaces using epoxide chemistry analogous to thereaction pathway described for the immobilization of polysaccharidesonto epoxidated surfaces in Li et al., Surface Modification of PolymericBiomaterials (B D Ratner and D G Castner, Eds), Plenum Press, NY, 1996pages 165-173 (the disclosure of which is incorporated herein in itsentirety), through isocyanate groups attached to the surface to producestable urethane linkages through thermal processes, or throughcarboxylic acid groups or their equivalents, such as acid chlorides, onthe surface to produce ester linkages. A furanone derivative thatcontains an aldehyde group can be linked onto surface amine groups usinga reductive amination reaction. A furanone derivative that contains acarboxylic acid group can be linked onto surface amine groups usingcarbodiimide chemistry. Other immobilization strategies are described inthe Examples below.

[0056] Interfacial coupling reactions must of course be selected notonly for their ability to achieve the desired covalent linkage but alsofor avoidance of adverse effects on the furanone compound(s) to beattached. Particularly, the furanone ring system tends to be labile toalkaline conditions. Such limitations are well known to those skilled inthe art. Among the many possible interfacial coupling reactions known inthe art, there is sufficient scope for selection of reactions thatproceed in a suitable pH range and with furanones substituted withvarious functional groups in various positions.

[0057] The wide range of interfacial reactions that can be used in thepresent invention enables the skilled practitioner to select one or morefuranone compounds for their particularly high effectiveness against thekey bacteria to be combated in a particular biomedical application (aswell as absence of adverse effects on the host system), and then designan interfacial linking reaction that can effectively produce a stablecoating of such furanone compounds. This is more advantageous thanhaving to select a furanone compound for its compatibility with aparticular manufacturing method.

[0058] Some solid substrate materials possess reactive surface chemicalgroups that can undergo chemical reactions with a partner group on afuranone molecule and thereby form a covalent interfacial linkagedirectly. Alternatively, in situ covalent linkage can be made directlythrough the addition of a doubly functionalised linker molecule to theactive surface in the presence of an appropriate furanone, or stepwiseby sequential addition of doubly functionalised linker molecules andthen an appropriate furanone. It is not always possible to immobilizefuranone compounds directly onto solid substrate materials; in thesecases, surface activation or one or more interfacial bonding layer(s) isused to effect covalent immobilization of the furanones. Such surfaceactivation is essential when immobilizing furanone compounds ontopolymeric materials such as fluoropolymers and polyolefins.

[0059] Surface activation of solid substrate materials can be achievedin a number of ways. Examples are corona discharge treatment or lowpressure plasma treatment of polymers. These methods are well known tointroduce a variety of functional groups onto polymeric surfaces.

[0060] An alternative approach is to provide an interfacial bondinglayer interspersed between the solid substrate material or biomedicaldevice and the furanone layer The application of a thin interfacialbonding layer can be done using methods such as dip coating, spincoating, or plasma polymerization. The chemistry of the bonding layer isselected such that appropriate reactive chemical groups are provided onthe surface of this layer, groups that then are accessible for reactionwith furanone molecules.

[0061] Particularly versatile is the subsequent application of multiplethin interfacial bonding layers; this method can provide a very widerange of desired chemical groups on the surface for the immobilizationof a wide range of functionalized furanones and enables usage offuranone compounds optimized for their biological efficacy.

[0062] The present invention, in its preferred forms, overcomes theshortcomings of the prior art. It provides surface-immobilized layerscomprising one or several chemical compounds from the class of compoundsknown as furanones or their analogs. One attractive feature of thisclass of compounds is that a substantial number of them are notcytotoxic to human or other mammalian cells and consequently can beselected to be biocompatible with a particular host environment, so thatadverse effects on adjacent host cells are eliminated whileantibacterial action is maintained. Moreover, by providing a thin,surface-coated layer of furanone compounds, the optical quality ofantibacterial devices of this invention is not reduced, which makes theinvention applicable to transparent ophthalmic devices such as contactlenses and intraocular lenses.

[0063] The present invention provides thin surface coatings that provideantimicrobial properties and/or antifungal properties to solid materialsonto which the coatings have been applied. More particularly, thecoatings may be designed to reduce or prevent colonization of biomedicaldevices by bacteria that cause adverse effects on the health of humanusers of biomedical devices when such devices are colonized by bacteria.

[0064] The active antibacterial layer comprises one or a plurality offuranone compounds selected for both their antibacterial activity andabsence of cytotoxicity as well as any other adverse biomedical effecton the host environment that the coated device contacts.

[0065] In order that the present invention may be more readilyunderstood, we provide the following non-limiting embodiments.

EMBODIMENTS OF THE INVENTION Comparative Example 1 (FEP)

[0066] Perfluorinated poly(ethylene-co-propylene) polymer (Teflon FEP,DuPont, 100A) in flat sheet form was washed carefully to remove loosesurface contamination. Analysis by X-ray photoelectron spectroscopy(XPS) (Kratos AXIS HSi, monochromatic excitation) demonstrated absenceof contaminants, which might interfere with bacterial assays. Theresults of elemental analysis of the surface using XPS are shown inTable 1.

Comparative Example 2 (FEP-HAPP)

[0067] A thin coating of a plasma polymer layer having surface aminegroups (HAPP) was deposited onto Teflon FEP sheet material fromplasma-activated heptylamine vapour as generally described in Griesserand Chatelier, Journal of Applied Polymer Science, Applied PolymerSymposium Vol. 46. Pages 361-384 (1990). Specifically, a piece of FEPsheet material of 12×60 mm is placed in a plasma deposition apparatus asdescribed in the above reference and the apparatus is evacuated to apressure of 10⁻³ mmHg. After establishing a constant flow of vapourevaporating off the monomer liquid n-heptylamine at a chamber pressureof 0.125 mmHg, the plasma deposition is carried out at a power of 20 Wand a frequency of 200 kHz. After 20 sec deposition time, the sample isremoved from the deposition apparatus and brought in contact with air.

[0068] XPS analyses confirmed successful deposition of this layer, byresults in accordance with the above publication The results ofelemental analysis of the surface using XPS are shown in Table 1. Thisamine-containing interfacial bonding layer was used for subsequentreactions as described below. The amine-containing bonding layer(FEP-HAPP) was also used as a control in bacterial adhesion andcolonization assays described below.

Comparative Example 3 (FEP-HAPP-PAAC)

[0069] Onto a sample of FEP coated with a heptylamine plasma polymerlayer as per the above example, a further interfacial bonding layer wasapplied by transferring the amine-coated FEP sample immediately afterplasma deposition to a flask containing 30 ml of a 0.1% aqueous solutionof poly(acrylic acid), MW 250,000 (Aldrich) at pH 4.0. Immediately afteradding the sample, 150 mg ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride EDC (Sigma)are dissolved in the solution. The sample was left at 4° C. underoccasional shaking for 16 h for reaction, following which it wasextensively washed with water to remove loosely bound (non-covalentlyadsorbed) poly(acrylic acid) (PAAC).

[0070] After rinsing, the presence of a covalently attached layer ofpolyacrylic acid was verified by XPS analysis, which showed the expectedcarboxyl contribution at 289 eV. The results of elemental analysis ofthe surface using XPS are shown in Table 1.

Comparative Example 4 (FEP-HAPP-PAAC-AZA)

[0071] Onto a carboxylated surface prepared as in Comparative Example 3,a layer of 4-azidoaniline was covalently attached by immersing a samplein 30 ml of an aqueous 2 mg/ml solution of 4-azidoaniline hydrochloride(Fluka) buffered to pH 8.8, which was prepared under darkroomconditions. Immediately after adding the poly(acrylic acid)-coated FEPsubstrate to the solution, 150 mg EDC is added to the solution. Afterincubating the sample for 16 h at 4° C. in the dark, the sample wastaken out of the solution, extensively washed with water and dried. Thiscreated an outermost surface possessing azide groups (AZA). Thesuccessful application of this azide layer was documented by theexpected increase in the nitrogen signal as measured by XPS. The resultsof elemental analysis of the surface using XPS are shown in Table 1.Samples coated thus were also used as a control in bacterial adhesionand colonisation assays.

Example 5 (FEP-HAPP-PAAC-AZA-furanone “24”)

[0072] Onto an azide surface prepared as in Comparative Example 4, afuranone derivative of the formula

[0073] (furanone “24”) was immobilized by reaction between the surfaceazide groups and the furanone compound under the following conditions;Still in the dark, the compound was evenly applied to the modified FEPsurface in a 200 mg/ml acetone solution. After complete evaporation ofthe acetone, the sample was irradiated for 12 min under a UV lamp at 40W/cm² and finally extensively washed with ethanol and air dried. Thefuranone-coated sample thus produced was analyzed by XPS to verifysuccessful coating. The results of elemental analysis of the surfaceusing XPS are shown in Table 1.

[0074] It is important to verify that the furanone compounds are indeedcovalently bonded to the underlying interfacial bonding layer, asopposed to adsorbed by physical forces, in which case at least a portionof the layer might be leached from the biomedical device andconsequently the desired antibacterial function would not be maintained.This was tested by performing the same set of operations but omittingthe UV light activation of azide reaction. After applying the furanonecompound under these conditions, it could be washed off completely, asattested by a Br signal close to background in XPS on the washed sample.

[0075] Samples coated thus (FEP-HAPP-PAAC-AZA-furanone “24”) weresubjected to bacterial adhesion and colonization assays as describedbelow.

Example 6 (FEP-HAPP-PAAC-AZA-furanone “4”)

[0076] The covalent immobilization of the furanone compound

[0077] to an FEP polymer substrate was carried out using the sameprocedures as in Example 5. XPS analysis for Br was used to ascertainsuccessful surface immobilization of the furanone compound. The resultsof elemental analysis of the surface using XPS are shown in Table 1.Samples coated thus were subjected to bacterial adhesion andcolonization assays as described below. TABLE 1 Sample % Si % F % C % O% N % Br Br/C FEP 0 66.2 33.5 0 0 0 0 (Comp. Example 1) FEP-HAPP 0 083.3 9.1 7.6 0 0 (Comp. Ex. 2) FEP-HAPP-PAAC 0 0 71.2 22.9 5.4 0 0(Comp. Ex. 3) FEP-HAPP-PAAC- 0 0 72.9 11.5 15.6 0 0 AZA (Comp. Ex. 4)FEP-HAPP-PAAC- 0 0.1 75.4 14.4 7.94 1.16 0.015 AZA-furanone “4” (Example6) FEP-EAPP-PAAC- 0.21 75.1 12.64 10.74 1.31 0.017 AZA-furanone “24”Example 5

Example 7 (FEP-HAPP-PAAC-AZA-furanone “22”)

[0078] The covalent immobilization of the compound (furanone “22”)

[0079] to an FEP polymer substrate was carried out using the sameprocedures as in Example 5. Samples coated thus were subjected tobacterial adhesion and colonization assays as described below.

Example 8 (FEP-HAPP-HMDI-furanone “4”)

[0080] The covalent immobilization of the furanone compound (furanone“4”)

[0081] to an FEP polymer substrate was carried out as follows: a sampleof FEP sheeting of 12×20 mm was placed in a plasma deposition apparatusand coated with a plasma polymer layer from the process vapourn-heptylamine at a pressure of 0.125 mmHg, at a power of 20 W, and afrequency of 200 kHz. After 20 s deposition time, the sample was removedfrom the plasma deposition apparatus and transferred to a containerholding 10 ml of a 10% (v/v) solution of hexamethylene diisocyanate HMDI(Aldrich) in acetonitrile. After incubation for 24 h at roomtemperature, the sample was washed extensively with acetonitrile andtransferred into 10 ml of a acetonitrile solution containing 0.2 ml ofthe furanone compound. After incubation for 72 h at room temperature,the sample was removed from the solution, washed extensively withacetonitrile, and finally air dried. XPS analysis for Br was used toascertain successful surface immobilization of the furanone compound.The results of elemental analysis of the surface using XPS are shown inTable 2. The samples thus coated were subjected to bacterial adhesionand colonization assays as described below. TABLE 2 Sample % Si % F % C% O % N % Br Br/C FEP 0 66.2 33.5 0 0 0 0 FEP-HAPP 0 0 83.3 9.1 76 0 0FEP-HAPP-HMDI 0.67 0 76.55 15.36 7.41 0 0 FEP-HAPP-HMDI- 0 0 75.26 14.095.7 4.86 0.065 furanone “4” covalent

[0082] Bacterial Adhesion and Colonization Assays

[0083] The bacterium used in the assays was Pseudomonas aeruginosastrain 6294. Bacterial cells were grown overnight at 35° C. intrypticase soy broth, harvested by centrifugation (3,000 g), washedthree times in phosphate buffered (pH 7.0) saline (PBS) and finallyresuspended in PBS to an optical density at 660 nm of 0.1. Bacterialcells (0.5 ml) were then added to the materials (with covalentlyattached furanones or without furanones as controls), and allowed toadhere at 35° C. for 10 min. After 10 min non-adhered or loosely adheredcells were removed by washing in PBS (3×1 ml). Materials were theneither analyzed for total number of bacterial cells adherent (initialadhesion), or alternatively, materials were transferred into 0.5 ml offresh TSB and incubated for 5 h at 35° C. incubator. After theincubation, the materials were analyzed for total numbers of bacterialcells (biofilm).

[0084] Materials used in the tests were: FEP-HAPP (control surface forHAPP-HMDI-coating), FEP-HAPP-HMDI-furanone “4”,FEP-HAPP-PAAC-AZA-furanone “4”, FEP-HAPP-PAAC-AZA (control surface forazide coating), FEP-HAPP-PAAC-AZA-furanone “24”,FEP-HAPP-PAAC-AZA-furanone “22”.

[0085] Bacterial adhesion was measured by staining the materials withcrystal violet. After rinsing off the stain, the number of bacteriaadherent to the surface was estimated by microscopic count. Results arepresented (Table 3) as percentage reduction compared to adhesion tocontrol (FEP-HAPP) in the initial adhesion and biofilm assays. TABLE 3Initial adhesion Biofilm formation FEP-HAPP-HMDI furanone 68% −16% “4”FEP-HAPP-PAAC-AZA- 47%   53% furanone “4” FEP-HAPP-PAAC-AZA- 42%   62%furanone “24” FEP-HAPP-PAAC-AZA- 35%   40% furanone “22”

[0086] Negative values indicate increase in adhesion/biofilm formationcompared to control.

Example 9

[0087] Attachment of Furanones to Cotton and Woollen Surfaces

[0088] The attachment method is based on a chemical (cyanuric chloride)which is used extensively in the dyestuffs industry for attachment ofone or two separate dyes to a fibre surface. This molecule has threeactive sites, one or two of which can be replaced by the dyestuff andthe remaining becomes a focus of attack by hydroxyl or amino-groupspresent in the fibre (e.g. cotton fibres or wool fibres).

[0089] A similar strategy as outlined below may be used for attachingfuranones to cotton and woollen surfaces. This involves reactingcyanuric chloride with one or two molecules of appropriate furanonefollowed by reaction with cotton or woollen fibres, which have freehydroxyl and amino groups respectively on their surface.

[0090] Throughout this specification the word “comprise”, or variationssuch as “comprises” or “comprising”, will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

[0091] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

1. A substrate having a plurality of immobilised furanone moietiesassociated with at least part of a surface of the substrate, wherein thefuranone moieties are derived from at least one compound selected from afuranone compound or an analog thereof.
 2. A substrate according toclaim 1, wherein furanone moieties are derived from at least twodifferent furanone compounds, at least two different furanone analogs ora combination of said furanone compounds and analogs.
 3. A substrateaccording to claim 1 or claim 2, wherein the furanone compound is anaturally occurring furanone.
 4. A substrate according to claim 1 orclaim 2 wherein the furanone compound is a synthetic furanone.
 5. Asubstrate according to any one of the previous claims wherein thefuranone compound is of the formula:

wherein R₁ is a moiety selected from the group consisting of H, halogen,formyl, carboxyl, cyano, ester, amide, alkyl, alkoxy, oxoalkyl, alkenyl,alkynyl, aryl or arylalkyl, which moiety may optionally be substitutedby one or more substituents; and/or interrupted by one or more heteroatoms; and/or straight chain, branched chain, hydrophobic, hydrophilicand/or fluorophilic; R₂, R₃ and R₄ are independently or all H orhalogen; and “═” represents either a double bond or a single bond.
 6. Asubstrate according to any one of the preceding claims, whereinimmobilisation on the surface is by means of covalent bonding.
 7. Asubstrate according to any one of claims 1 to 5, wherein immobilisationon the surface is by chemical bonding other than covalent bonding.
 8. Asubstrate according to any one of the previous claims, wherein thesurface of the substrate has been subjected to surface activation priorto immobilisation of the furanone moieties.
 9. A substrate according toany one of the preceding claims wherein one or more interfacial bondinglayers are provided on the surface of the substrate to which are bondedthe furanone moieties, the interfacial layer(s) optionally subjected tosurface activation prior to immobilisation of the furanone moieties. 10.A substrate according to claim 9, wherein the interfacial bondinglayer(s) is (are) applied to a surface of the substrate by a methodselected from the group consisting of dip coating, spin coating,spraying and plasma polymerisation.
 11. A substrate according to any oneof the preceding claims wherein the material of the substrate isselected from at least one of the group consisting of metals, ceramics,glasses, natural polymers, synthetic polymers and natural materials. 12.A substrate according to claim 11, wherein the material is selected fromthe group consisting of noble metals, titanium, steel, hydroxyapatite,ethylene polymers and copolymers, polyurethanes, organosiloxanes,perfluorinated polymers, acrylic hydrogel polymers and copolymers,siloxane hydrogel polymers and copolymers and natural and syntheticelastomers.
 13. A substrate according to claim 12 wherein the materialis non-functionalised or contains free functional groups forimmobilisation.
 14. A substrate according to claim 11, wherein thenatural material is selected from the group consisting of seeds, grains,seed products, fibres, wool, hair, silk, cotton, chitin, collagen,animal organs and animal hides.
 15. A substrate according to any one ofthe preceding claims, wherein the substrate is solid, semi-solid, rigidor flexible.
 16. A substrate according to any one of the precedingclaims, which is shaped.
 17. A substrate according to any one of claims1 to 15, which is unshaped.
 18. A substrate according to any one of thepreceding claims in the form of a natural or synthetic filament orfibre.
 19. A substrate according to claim 18, wherein the fibre is anatural fibre selected from wool, cotton and hemp.
 20. A substrateaccording to any one of the preceding claims in the form of a woven ornon-woven film, sheet or textile.
 21. A substrate according any one ofthe preceding claims, which is formed from a material that is suitablefor use in a biomedical application.
 22. An article consisting of orcomprising a substrate in accordance with any one of the precedingclaims.
 23. An article according to claim 22, which is a biomedicaldevice.
 24. An article according to claim 23, wherein the biomedicaldevice is an implantable device.
 25. An article according to claim 24,wherein the biomedical device is selected from the group consisting ofcatheters, stents, orthopaedic implants, soft tissue augmentationdevices, blood contacting devices, heart valves, arteries, veins,pacemakers, ear implant devices, electrodes and dialysis devices.
 26. Anarticle according to claim 22, which is a contact lens or intraocularlens.
 27. An article according to claim 22, which is a personal hygieneproduct.
 28. An article according to claim 22, which is a food containeror food wrapping.
 29. An article according to claim 22 which is anarticle of clothing, headware or footware or a component thereof.
 30. Amethod of forming an antimicrobial and/or antifungal layer on asubstrate including: (a) providing a substrate; (b) providing at leastone furanone compound or an analog thereof; (c) optionally treating atleast part of the surface of the substrate to activate the surface; (d)reacting the at least one furanone compound or an analog thereof withthe optionally treated surface to immobilize the at least one furanoneor analog thereof.